Serum of unimmunized mice contains an inhibitor capable of suppressing the immune response of mouse spleen cell cultures to sheep erythrocytes. The concentration of this serum inhibitor increases after immunization of mice with erythrocyte antigens. The serum inhibitor exerted its effect in the early stages of the immune response. Addition of mouse serum to spleen cells up to 48 hr after initiation of in vitro cultures was immunosuppressive. Washing or trypsinization of serum-incubated spleen cells removed the inhibitory effect. Plastic-adherent populations of spleen cells were not inactivated by the mouse serum factor whereas nonadherent cells were markedly affected. The inhibitory factor was purified by ammonium sulfate precipitation and Sephadex chromatography. The immunosuppressive factor was found to migrate to the α-region, and its inhibitory function was not related to RNase activity.